WO2006005887A1

WO2006005887A1 - Method for localizing a chemical or biological species on a substrate, analyzing microsystem and biochip

Info

Publication number: WO2006005887A1
Application number: PCT/FR2005/050543
Authority: WO
Inventors: Marc Plissonnier; Isabelle Chartier; Mathias Borella; Nicolas Glade
Original assignee: Commissariat A L'energie Atomique
Priority date: 2004-07-07
Filing date: 2005-07-05
Publication date: 2006-01-19
Also published as: FR2872911A1; FR2872911B1

Abstract

The invention concerns a method for localizing a chemical or biological species on a substrate, a textured substrate (1) and an analyzing microsystem and a biochip. The method includes the following steps: providing a substrate (S); depositing on at least one surface of the substrate a layer of a material of formula (a) SiOxCy:H wherein 0,7 ≤ x ≤ 1,3 and 1,5 ≤ y ≤ 2; decomposing by ultraviolet treatment at least one localized zone of the material of formula (a) deposited in a material of formula (ß) SiOx1Cy1:H wherein 1,7 ≤ x1 ≤ 2,5 and 0,1 ≤ y1 ≤ 0,4; and contacting said localized zones of the material of formula (ß) with a solution of the chemical or biological species.

Description

METHOD FOR LOCATING A CHEMICAL OR BIOLOGICAL SPECIES ON A SUBSTRATE, MICROSYSTEM FOR ANALYSIS AND

BIOCHIP

DESCRIPTION

TECHNICAL AREA

The present invention relates to a method for locating a chemical or biological species on a substrate, to a textured substrate that can be obtained from this method, as well as to a micro-analysis system and a biochip capable of to be obtained from this process. The present invention finds particular applications in the manufacture of substrates serving as supports for tests in chemistry or biology, in particular where substrate surfaces are structured locally to obtain "zones" ("spots" in English) where elements chemical or biological substances can adhere and / or develop for their analysis and / or detection.

STATE OF THE PRIOR ART

The known methods for locating chemical species on a substrate are neither simple nor very high selectivity: the main problem is that cells, molecules and liquids adhere everywhere.

The micropatterning of biological species, that is to say the control of the fixation and non-fixation of chemical or biological cells or molecules on Precise areas of a surface is usually obtained following two different techniques which are photolithography and printing.

The technique of micropatterning by photolithography has already been widely described, as for example in document [1] EP-A-I 260 863, "Micropatterning of plasma polymerized polymer", Winther-Jensen Bjoern; Bouaidat Salim; Jonsmann Jacques. It consists of forming cavities in an organic or inorganic material using the technologies used in the microelectronics industry. However, this technology has several disadvantages. In particular, photolithography requires the use of solvents and bases that are incompatible with many biological molecules. The equipment needed to implement this technology is also complex and relatively expensive for biological applications.

Printing ("printing" in English) by micro-contact ("micro stamping" in English) is another technique that has been used to achieve the "micropatterning" of biological species on substrate surfaces. Using this technique, inorganic substrates such as glass and silicon have been micro-matrixed by depositing inks on their surface. In this technique, proteins were applied to a dense networked surface from a pre-coated poly (dimethylsiloxane) pad. Other methods use the localized modification of the polarity of the surface by applying a specific buffer, that is to say micromatricked, and coated of product that can locally change the polarity of the substrate. [2] US Patent No. 20030104614, "Micropatterning surfaces of polymeric substrate", Schmalenberg Kristine (US); Buettner Helen M. (US); Uhrich Kathryn describes such a technique.

Unfortunately, in the first case, the active biochemical molecules are in contact with foreign constituents that can contaminate them and modify their behavior. In the second case, the localized modification of the polarity must be localized just before the deposit of the biochemical molecules. This technique is also potentially sensitive to cleaning. These two techniques are furthermore based on the deposition of a film intended to increase the fixation of the biological molecules in certain zones or of a film blocking the fixation of the biological molecules in complementary zones, and in all cases the film does not is not continuous on the substrate which weakens the mechanical strength of the deposit (aging and held cleaning). Deposition of this or these films also requires equipment and apparatus, which extends the manufacturing time of the substrate and increases the cost of its manufacture. Thus, in the field of the manufacture of substrates serving as supports for tests in chemistry or biology, there is a real need for new manufacturing processes which provide a solution to the aforementioned drawbacks, and which also make it possible to work on scales which are always the

more reduced as improvements are made to microsystems analysis and biochips.

DISCLOSURE OF THE INVENTION The present invention specifically provides a novel method for producing textured substrates that can be used as supports for tests in chemistry or biology, which notably provides a solution to the aforementioned drawbacks of the prior art, which is easy to implement, fast and presenting a reduced cost.

The method of the invention is a method of locating a chemical or biological species on a substrate comprising the following steps:

(a) providing a substrate; (b) depositing on at least one surface of the substrate a layer of a material of formula (α) SiO _x C _y : H where 0.7 <x <1.3 and 1.5 <y <2;

(c) modified by ultraviolet or plasma treatment at least one localized zone of the layer of material of formula (α) deposited in step (b) in a material of formula (β) SiO _x iC _y i: H where 1, 7 ≤ xl ≤ 2.5 and 0.1 <yl <0.4; and

(d) contacting said localized areas of the material of formula (β) with a solution of the chemical or biological species.

The present invention also relates to a textured substrate comprising: a material of formula (α) SiO _x Cy: H where 0.7 <x <1.3 and 1.5 <y <2; and a material of formula (β) SiO _x iC _y i: H where

1.7 <xl <2.5 and 0.1 <yl <0.4. This substrate may further comprise, especially during its use, but also for marketing, a chemical or biological species related to the material of formula (β). The invention relates to the forging, or texturing, of substrate surfaces, and in particular the micromatrixing of chemical or biological species on substrate surfaces.

The substrate is in fact the support on which the material layer (α) is deposited in step (b) of the process. It may be any solid material known to those skilled in the art, such as, for example, the support materials used for the manufacture of analytical microsystems and biochips. It can be an organic or inorganic substrate. It may consist for example of a material selected from the group consisting of glass, silica, polycarbonate, polymethyl methacrylate (PMMA), polystyrene, cycloolefin copolymer (COC), and polystyrene acrylonitrile (SAN).

The substrate may or may not be flat, that is to say: comprise microstructures (channels, cavities, different levels, etc.) producing a microsystem (fluidic, mechanical, optical such as electromechanical microsystems (MEMS) or optoelectromechanical microsystems (MOEMS)); have electrical functions, for example by metal deposition for electrodes; (CMOS circuitry for complementary metal oxide semi conductor), optical (deposition of thin layers adapted to make mirrors, anti-reflective zones, waveguides, etc.).

Before step (b) of the method of the invention, the substrate may be cleaned in order to improve the adhesion of the material layer (α) to its surface. This cleaning can be for example a chemical cleaning, possibly followed by a heat treatment. Cleaning techniques, in particular those used for the preparation of biochip carriers, known to those skilled in the art are usable. For this cleaning, any solvent suitable for dusting and / or degreasing the surface of the substrate may be used, preferably without damaging it. Trichlorethylene, acetone, ethyl alcohol, deionized water, acidic or basic solutions, etc. may be mentioned as cleaning solvents, for example. The cleaning may consist of bathing the substrate in one or more of these solvents successively. Cleaning is usually followed by drying. The cleaning may also simply consist of ridding the substrate of its dust with a jet of compressed air.

The step (b) of depositing on at least one surface of the substrate a layer of a material of formula (α) may be carried out by any technique known to those skilled in the art to deposit this type of material on a surface. It may be for example a plasma-assisted chemical vapor deposition

(PECVD deposit) of a polysiloxane (or organosilane). The polysiloxane is a precursor of the material (α) which the

transforms into said material (α) during its deposition on the surface of the substrate.

The polysiloxane may be chosen for example from hexamethyldisiloxane (HMDSO) and octamethylcyclotetrasiloxane (OMCTS), tetramethylcyclotetrasiloxane (TMCTS).

The use of plasma chemical vapor deposition (PECVD or PACVD) technology results in very thin deposition thicknesses that can be less than 50 nanometers, and even up to 5-10 nm, which guarantees excellent adhesion of the deposit on any type of substrate. A PECVD or PACVD technique that can be used to implement the present invention is described for example in document [3] Svend S. Eskildsen et al.

"CVD Plasma: Process Capabilities and Economics Aspects", Surface and Coatings Technology, 116-119

(1999) 18-24. This material of formula (α) has the particularity of being amorphous. According to the invention, the material layer of formula (α) may be deposited on said surface of the substrate at a thickness of 5 nm to 5 μm. In fact, it is easy to understand that the minimum thickness is a thickness which advantageously makes it possible to cover the at least one surface of the substrate without leaving holes. The maximum thickness is not limited, it is however superfluous to use too large amounts of material to implement the present invention. The step (c) of modification is carried out by localized treatment. "Localized" means in this "defined". In this step, a defined zone, in thickness and on the surface, of the material layer of formula (α) is converted into a material of formula (β). The size of the surface of the transformed material is determined essentially by the surface of the material exposed to ultraviolet radiation or plasma.

The ultraviolet treatment or the plasma treatment may be any treatment known to those skilled in the art which induces the transformation of the material of formula (α) into a material of formula (β). UV treatment can also be performed by laser. The treatment is advantageously carried out by irradiation-type treatment (for example UVO: ultraviolet-ozone - ozone is created by UV) or by plasma-type treatment (for example helium, argon, corona, atmospheric plasma). , SF ₆ / C> 2, CH 3 O 2, etc.). When the treatment is a light irradiation, the wavelength is generally less than 300 nm (threshold at which the molecular oxygen can be converted into ozone), in particular equal to 184.9 nm and 253.7 nm. The ultraviolet can be generated by any means known to those skilled in the art, for example by a low pressure mercury vapor lamp for treatment in the presence of ozone. The ultraviolet treatment can be carried out advantageously in the presence of oxygen.

The duration of this treatment is determined in particular by the operating conditions used and by the desired decomposition thickness. For layers of materials (α) such as those mentioned above, a treatment time of 1 to 20 minutes is usually sufficient.

The surface location of the zones where the material (α) is converted can be obtained for example by positioning an absorbent mask around said at least one zone during the treatment. It may be a mask of the mechanical type, for example in the form of a cover, or simply optical in the case of ultraviolet radiation, used where the material (α) must not be modified. This mask has for example one or openings (s) localising the material zone (s) (α) that will be (are) modified (s). Advantageously, localized areas of material (β) are obtained without using photolithography or micro-contact techniques. The mask may consist of a material chosen from a metallic material (stainless steel, steel, etc.), carbon, etc.

The method of the invention therefore consists in depositing an SiO _x Cy: H type amorphous material on a surface of a substrate, the composition of which is 0.7 <x <1.3 and 1.5 <y <2 (or the composition domain) is determined so that, in particular, chemical or biological species, for example living cells, can not adhere to its surface. This material is then modified locally to obtain a material of SiO _x iC _y i: H such that 1.7 <Xi <2.5 and 0.1 <yi <0.4 allowing the adhesion of chemical and / or biological species . This new composition allows for example a normal development of living cells and can be used for biochemical analysis applications. the

The object of the invention is therefore to enable the localization of chemical or biological species by promoting their adhesion on specific areas of the surface of a substrate. Localization is further facilitated by a modification of the surface energy, the material passing from a hydrophobic character (material (α)) to hydrophilic (material (β)).

According to the invention, the material (β) may also be functionalized for the attachment thereto of a chemical or biological species. Also, the present invention also relates to a substrate according to the invention, in which the material of formula

(β) is thus functionalized. The functionalization is carried out by any chemical means known to those skilled in the art in order to fix molecules on a silica-based surface. It may be for example a functionalization intended to form silanol groups, for example for the purpose of fixing nucleic acids. These techniques are well known to those skilled in the art.

According to the invention, step (d) can be carried out for example by immersing the substrate obtained in step (c) in said solution of the chemical or biological species, or by depositing in the form of a drop of said solution of the biological species on said localized zone of material (β) obtained in step (c). The immersion is preferably made for a time sufficient to allow the adhesion of chemical or biological species, for example cells. The deposition in the form of a drop can be carried out for example by the techniques known to those skilled in the art for disposing drops of a solution on the surface of a substrate, for example by micropipettage or by means of an ink jet type apparatus ("jet printing" in English). The solution may be any suitable solution to contain said chemical species. Preferably, it is not corrosive for the substrate of the present invention, in particular for the materials (α) and (β). It may be for example an aqueous solution, for example also a physiological liquid, a buffer solution, a biological buffer, etc. It can also be a solution of polymers (Nafion (trademark), glues such as epoxy glues, conductive glues, resins), oils, organic or inorganic solvents, organic or inorganic acids, etc. .

This step (d) can be performed without functionalization, directly on the substrate of the present invention. It can also be performed after functionalization.

According to the invention, when the species is biological, it can be chosen for example from the group consisting of a eukaryotic cell, a prokaryotic cell, a protein, a glycoprotein, an enzyme, a an antigen, an antibody, DNA, RNA, a hormone, a living tissue, a protein or glycoprotein network.

According to the invention, when the species is chemical, it may be a polymer, for example organic. The inventors of the present have in fact unexpectedly noticed that the material zones (β) are very favorable to the adhesion and growth of cells and the material zones (α) are very unfavorable to their growth. The present invention thus makes it possible to locate the adhesion of living cells.

All the photolithography and etching technologies currently used do not make it possible to obtain such behavior. In addition, on the substrates obtained by the techniques of the prior art, the cells adhere and often develop indifferently outside the zones defined by these techniques. This is not the case in the present invention.

The micromatrixing of biological species on a surface finds a number of applications such as combinatorial screening strategies, multiple analytical detectors and the ability to modulate cell-substrate interactions by spatially directing cell growth.

The stamping obtained by the process of the present invention is stable over time and is not sensitive to different chemical cleanings. In addition, the technique used in the present invention is insensitive to the aging of the substrate.

The method of the present invention advantageously makes it possible to process non-horizontal planes and thus substrates structured by fluidic micro-channels for example. The location of "hydrophilic" zones materialized by the localized zones of material (β) makes it possible to confer in addition to the substrate fluidic properties: it allows a localization of drops of culture liquid in the zones adhering to the biological molecules, filling or definition of capillaries.

Deposition of chemical or biological molecules is easy because the non-adherent zones (material of formula (α)) are hydrophobic and those adherent (material of formula (β)) are hydrophilic. The solutions of the deposited chemical or biological species are therefore very precisely located.

There is no lithography or deposition of surface structuring compounds, such as resins. The film is continuous and conforms to possible structuring of the substrate. The adhesion properties can thus be imparted to non-horizontal surfaces. The method of the present invention can be applied over large areas, allowing the collective fabrication of devices. The method of the invention is also economically very profitable since it is very simple to achieve. Another advantage of the present invention is that the SiOxCy: H material is transparent in X-ray fluorescence. It is therefore possible by X-ray fluorescence, for example below a glass slide as a substrate, to visualize the labeled cells or not. The present invention can be used for example for the manufacture of a biochip or a microsystem of chemical or biochemical analysis. The present invention thus also relates to a microsystem for chemical or biochemical analysis, or to a biochip, comprising a substrate according to the invention. Indeed, once the chemical or biological species are bonded to the surface of the substrate according to the present invention, they can be analyzed according to the methods known to those skilled in the art for the analysis and / or the detection of molecules or cells on the surface of a substrate, particularly in the field of biochips.

Other possible applications of the process of the invention are, for example, the location of glue (which represents the chemical species in the process of the invention) on a hydrophilic zone, for example for bonding a hood; the location of liquid drops on localized areas of a surface of a substrate for electrically connecting two surfaces, for example superimposed, for example in a "flip chip" method or to form a conventional connection by locating conductive tracks; locating magnetic elements for a localized deposition of magnets after evaporation of a drop of solution comprising said magnets; depositing on localized areas a surface of a droplet substrate forming microlenses or depositing on localized areas a surface of a liquid crystal substrate in the field of optics.

For the implementation of these other applications, according to the invention, the protocols described in the US Pat. following documents by replacing the substrate described in these documents by that of the present invention. In other words, the localized areas of material of formula (β) of the present invention serve as supports for carrying out the processes described in the following documents:

glue localization: for example bonding of a cover (for example seal for bonnet sealing): WO-A-04/043849 (deposit of silicone material); glue localization: for example bonding of microstructured substrates (glue deposition): FR-A-

2,856,047; locating drops of liquid for electrically connecting two surfaces (for example hybridization "flip chip" - for example deposition of conductive material): FR-A-2,748,849;

depositing drops forming microlenses or deposition of liquid crystals in the field of optics (for example liquid lenses - deposition of liquid on a substrate): FR-A-2 851 052.

Other characteristics will become apparent upon reading the examples which follow, given by way of illustration and without limitation, with reference to the appended figures.

Brief description of the figures

Figure 1: Schematic representation of a textured substrate obtained according to the method of the present invention. - Figure 2: graphical representation of the number of cells according to the incubation time for three different samples, comprising all three a layer of material of formula (α) (precursor OMCTS), and each different durations of localized changes of material of formula (β) by ultraviolet + ozone treatment (UVO). In this figure, N = number of cells counted per ml (see § E below).

3: graphical representation of the comparison of the number of cells after 96 hours of culture for OMCTS / OMCOS UVO 10 minutes and polycarbonate / polycarbonate + UVO 10 minutes.

Figure 4: Photograph showing the localized adhesion of living cells on a textured substrate according to the invention.

EXAMPLES

A- preparation of the substrate

The support which has been used to implement the method of the invention is a glass slide.

The blade is freed from its dust by a jet of compressed air.

B-deposit of a layer of material of formula (α) The blade is introduced into a PECVD equipment to deposit SiO0.7C0.05H0.25 •

The deposition conditions are as follows: capacitive coupling plasma OMCTSO (Aldrich, 98%) / He

(99.999%). RF power: 50 watt (0.1 watt / cm ² , advanced energy RFlOS power generator), pressure: 0.2 mBar, OMCTS flow rate = 2.82x10 ^-4 mole / min, He: 500 standard cm ³ , interelectrode distance: 30 mm, temperature: ambient.

A layer of SiO0.7C0.05H0.25 of 50 nm was deposited.

C- Modification of material of formula (α) in material of formula (β) by ultraviolet treatment + ozone

Three identical samples are treated in different ways. The three samples comprise on their surface a layer of material of formula (α) (OMCTS precursor) deposited according to the method described in paragraph B.

The localized modification of the material of formula (α) in material of formula (β) was carried out by ultraviolet (UVO) treatment (photochemical modification).

In this example the localized modification is carried out by a UV-Ozone cleaner (UVO Cleaner) model 342-220 manufactured by Jelight Company (2 Mason, Irvine, CA92618 U.S.A.).

The apparatus operates in ambient air, that is, at atmospheric pressure and ambient temperature. A low-pressure mercury vapor lamp, a grid-shaped, emitting a wide ultraviolet radiation, among others bands in the deep UV at 184.9 nm and 253.7 nm was used. The lamp has a power of 28 μW / cm ² to 6 mm.

The temperature increases by a few tens of degrees during the treatment (up to about 50-70 °) for a long exposure time (greater than 20 min) but does not alter, a priori, the process. The lamp of the device is put to preheat 10 minutes to obtain an optimal UV emission.

The substrate is placed in a sample holder location at a distance of approximately 5 mm to 1 cm from the mercury lamp (as recommended in the device manual).

In the various tests carried out, the exposure is greater than 1 minute and can last several tens of minutes depending on the conditions: - if the surface is directly exposed to

UV, through a mechanical mask for example, the exposure is of the order of 1 to 10 minutes; on the other hand, through a quartz optical mask, for example 2 mm thick, which absorbs a good amount of deep UV: about 50% loss at 184.9 nm and 20% loss at 253.7 nm, it takes a exposure from 30 minutes to 2 hours to obtain an equivalent effect (the problem is related to the poor transmittance of quartz in the deep UV). The location of the irradiated area is achieved using a mechanical steel mask. The mechanical mask is kept pressed on the blade by a pinch on the sides, leaving the pattern accessible

(holes) to UV. Different modification treatment times, from one sample to the next, were performed: sample 1: OMCTS + UVO 3 minutes sample 2: OMCTS + UVO 5 minutes - sample 3: OMCTS + UVO 10 minutes A control sample is also performed: OMCTS without modification treatment.

The appended FIG. 1 is a schematic representation of a textured substrate (1) obtained according to the method of the present invention. In this figure, the reference (S) indicates the support of silicon, (α) indicates the material of formula (α) (OMCTS), and (β) indicates the areas where the material of formula (β) has been formed by modification. photochemical material type (α).

D-decomposition of material of formula (α) in material of formula (β) by He plasma treatment

He plasma treatment makes it possible to obtain equivalent or slightly better results.

In this case the protocol is the following, instead of the protocol of paragraph C above: the mechanical mask is maintained plated on the substrate by a pinch on the sides, leaving the pattern (holes) to the plasma. The substrate is introduced into the PECVD equipment described above and is then directly exposed to the plasma.

The plasma treatment Helium lasts 1 minute, the conditions are RF 200W, 0.2 mbar, 500 sccm He.

E- bringing the material of formula (β) into contact with a biological species

Live hamster ovary (CHO) cells were put in aqueous solution at 7 × 10 ³ cells / ml. The substrates of the different samples obtained according to the protocols of paragraphs C and D were immersed in the solution.

The textured substrates thus covered with drops of solution of living cells were then incubated for 0, 24, 48, 72 and 96 hours at 37 ^° C.

The number of adherent cells on the aforementioned zones was counted by manual counting either directly under the microscope or on microscope slides at incubation times of 0, 24, 48, 72 and 96 hours, after washing the samples by buffer. physiological.

The appended FIG. 2 shows the results of the measurements for the various samples having undergone a decomposition treatment of 3, 5 and 10 minutes.

It is noted in this figure that the difference in the number of cells between the composition zone SiO _x C _y H ₂ and the composition zone SiO _x iC _y iH _z i is very important (factor 10) after 96 hours of incubation. These results clearly show the difference in adhesion of cells to a substrate by the method of the present invention.

Optimal cell spreading

(31 μm +/- 17 μm) is obtained with 3 to 5 minutes of UVO treatment with a mechanical mask. With a longer treatment, the cells spread much less. Cell adhesion is very sensitive to the presence of charges and hydrogen bonds. In the case of OMCTS substrates treated with UVO, the observed optimal behavior is obtained for treatments of 3 to 7 minutes, that is to say when the proportion of hydrogen bonds is high among the polar interactions. Below, the polar interactions are too weak to facilitate adhesion. Above this time, the number of charges becomes very important. These charges, a priori negative, will tend to induce an electrostatic repulsion of the cells (negatively charged on their surface) which strongly opposes the adhesion induced by the hydrogen bonds. Figure 4 is a photograph showing the localized adhesion of the cells in 480 μm diameter areas after 72 hours of culture and a simple washing of non-adherent cells to the saline buffer.

F- Comparative

The attached FIG. 3 compares adherent cell count results on the surface of four different substrates: a surface obtained from a polycarbonate support treated according to the protocols described in paragraphs (B) and (E) (no modification processing) above (OMTCS) ("AA" for prior art); a surface obtained from a polycarbonate support treated according to the process of the invention described in paragraphs (B) to (E) above (10 minute decomposition treatment) (OMTCS + UVO 10 minutes) ( α and β materials); a polycarbonate (PC) surface not treated by the process of the invention. a polycarbonate surface subjected only to the decomposition treatment of 10 minutes (no layer of material (α)) (PC + UVO 10 minutes).

The protocol of paragraph D has been applied for depositing living CHO cells. Cells were counted after 96 hours of incubation.

The number of cells before and after UV + ozone treatment does not make it possible to obtain a very large cell growth difference on the polycarbonate surfaces (AA results in FIG. 3) which have not been treated according to the method of the invention. present invention.

On the other hand, on the surface treated by the process of the invention, a clear difference in adhesion occurs between the localized zones treated photochemically (material of formula (α)) and the untreated zones (material of formula (β)).

Similar results are obtained with a cycloolefin copolymer (COC) or polystyrene acrylonitrile (SAN) support.

On Helium Plasma treated substrates, the cells multiply as fast but spread much more than UVO-treated substrates. The cells have a measurable spread by their mean diameter of 42 μm +/- 15 μm on the substrates treated plasma Helium, against 31 μm +/- 17 μm on a treated substrate UVO 3 minutes, and 20 μm +/- 3 μm on an OMTCS substrate without treatment. 6- Application of the present invention on another support

In this example, the glass support used in Examples A to D above is replaced by another material and the method of the invention is applied as in Examples A to D.

It is shown in this example, in particular, that the method of the invention operates on any type of support, and provides other advantages that those mentioned above related to the characteristics of the substrate of the present invention.

The support chosen for the implementation of the present invention is the electrode of a fuel cell, in particular batteries described in EP-B1-241432, US-A-4,877,694 and US-A-4. In fact, the hydrophilic layers described in these documents are replaced by the material β obtained from the material α according to the method of the invention.

As a reminder, fuel cells comprise two electrodes in which occur the reactions of reduction of oxygen and oxidation of a fuel which may be a gas or a liquid. These electrodes generally consist of several layers. A first fabric or carbon felt backing layer impregnated with a fluorophore or derivative polymer having a strongly hydrophobic character to which is added a diffusion layer composed of a mixture of carbon powder and fluoropolymer. Finally, the last layer called active layer is composed of carbon, a catalyst and a polymeric binder. The nature of the oxygen reduction reactions produces water at the catalytic sites. Consequently, the commercial electrodes use a fluoropolymer binder whose hydrophobic nature makes it possible to repel the water produced, thus avoiding the flooding of the "cathodes". If the water is not removed: the cathode is flooded, which prevents oxygen from accessing the catalytic sites.

The supply of liquid fuel or reactive ions to the electrodes, however, requires a hydrophilic nature of the materials used. In the case where the batteries use a liquid fuel such as methanol, ethylene glycol or sodium borohydride in solution, the hydrophilic nature of the electrode facilitates its transport to the catalytic sites. Commercially available electrodes having a hydrophobic nature limit this transport, which causes the performance of the battery to fall. The surface energy of commercial electrodes is very low, not very polar. They have little affinity with water that wets the substrate very little (contact angle measured: 128.8 °).

Another limitation related to commercial electrodes of hydrophobic character lies in the drying of fuel cells (for example PEMFC batteries for "proton methanol fuel cell") using hydrogen. If the latter are quite suitable for evacuating water when the current density is high, they are less at low current density or on the contrary, it is necessary the the

keep the water formed to avoid drying out the cathode and the membrane.

The management of water and the supply of catalytic reactant sites is a major factor in fuel cells. Therefore, the amount of binder conditions the efficiency of the cathode. Optimizing the amount of binder requires many tests. Indeed, it can coat platinum sites and therefore reduce the number of "active" sites if it is used in too large quantities. Similarly, if used sparingly, electrolyte will drown the "active" sites that will then no longer have access to oxygen. The balance to be achieved between the electrolyte access and the oxygen and the access to the electrons is an essential data of operation of the electrodes.

For example in EP-B-00241 432, the authors describe an air cathode whose active layer contains hydrophobic and hydrophilic materials, the gas diffusion layer being hydrophobic. This document describes the superposition of hydrophilic (fiber-based) and hydrophobic parts in the form of powder. They are assembled in the form of sheets to form the active layer. In a slightly different way, in US-A-

The general composition of the cathodes is based on two layers, one hydrophobic (gaseous diffusion layer) and the other hydrophilic (active layer); it is replaced by a "dual" layer containing active materials and gas diffusion. the the

The hydrophilic nature is enhanced by the use of hydrophilic polyesters.

In US-A-4,615,954 the gas electrolyte permeability balance is ensured by a particular mixture of components comprising a very high surface area carbon.

The methods described in these three documents have the following drawbacks:

- Production of complex layers requiring assembly of several hydrophobic and hydrophilic layers. With a risk of separation of the layers between them or formation of an electrolyte tight zone located at the junction between two layers. - Addition of hydrophobic or hydrophilic compounds in the form of powders or use of high developed surface material decreasing the intrinsic cohesion of the electrode, which promotes delamination and therefore a decrease in performance.

use of two different polymers each having a hydrophilic or hydrophobic character and the implementation of which requires an additional homogeneous mixing step. As shown in this example, the present invention solves these three disadvantages simultaneously.

In order to maintain a balance between the liquid electrolyte permeability and the reactive gas, the inventors modify, thanks to the method of the present invention, the surface energy of the electrodes. the the the

The present invention makes it possible to render the treated surfaces super hydrophilic. This allows on the one hand to ensure the transport of polar fuels to the catalytic sites and thus increase the electrochemical performance of fuel cells (for example of the DMFC (direct methanol fuel cell), DEFC, PAFC, SAFC, etc.) and on the other hand to limit the drying of proton fuel cells (for example PEMFC type) when the latter work at high voltage (high efficiency). Thus, the method of the invention makes it possible to improve the stability of their performance.

The inventors have modified one of the faces of the cathode to make it more hydrophilic according to the protocol described in the previous examples. This made it possible to promote electrolyte access to the reactive sites of the cathode without limiting the diffusion of oxygen from the untreated side. The use of hydrophobic halogenated polymers in sufficient quantity makes it possible both to guarantee the hydrophobicity of the cathode and the cohesion thereof.

The modification is generated by plasma treatment or ultrathin deposition of a superhydrophilic layer according to the present invention. This treatment is applied only on the face of the electrode in contact with the electrolyte or the liquid fuel. The diffusion of electrolyte or fuel in the cathode is then improved to a small thickness. Tests carried out in this example have validated the modification of the penetration of

The electrolyte on a cathode since a helium plasma treatment has made it possible to modify the contact angle (solid electrolyte interface) of 128 to 26 °.

Likewise, with an oxygen plasma, the measured contact angle is then 36.2 °.

Plasma deposition of superhydrophilic material (SiO _x ) according to the present invention has made it possible to validate a total wetting.

Polarization tests have shown an improved current density of 3% for helium plasma treatment.

The present invention therefore also relates to a fuel cell comprising an electrolyte and electrodes, wherein the wettability of at least one of the electrodes is modified by a method comprising the following steps:

(a) providing an electrode that serves as a substrate;

(b) depositing on at least one surface of the substrate a layer of a material of formula (α) SiO _x C _y : H where 0.7 <x <1.3 and 1.5 <y <2;

(c) modifying by ultraviolet or plasma treatment at least one localized zone of the layer of material of formula (α) deposited in step (b) in a material of formula (β) SiO _x iC _y i: H where 1, 7 ≤ xl ≤ 2.5 and 0.1 <yl <0.4.

According to the invention, the cathode or the anode may therefore have been treated with plasma, for example with helium or oxygen plasma. All the the

The features of this process are already described in the "disclosure" portion of the present invention. The fuel cells may be all those known to those skilled in the art, for example those described in the aforementioned documents.

Thus, this example demonstrates the following advantages of the method of the invention:

Increase in the number of accessible catalytic sites, allowing an increase in the current density of the electrode;

Use of cathodes with a high binder content (20 to 70%) which allows a better cohesion improving the lifetime of the electrode. Limitation of the number of polymers used; the choice of the plasma or deposition technique makes it possible, by modifying the parameters, to adjust the equilibrium of electrolyte permeability at different binder levels (from 10 to 70%).

Claims

1. Substrate comprising: a material of formula (α) SiO _x C _y : H where 0.7 ≤ x ≤ 1.3 and 1.5 ≤ y ≤ 2; and a material of formula (β) SiO _x iC _y i: H where 1.7 <xl <2.5 and 0.1 <yl <0.4.

2. Substrate according to claim 1, said substrate further comprising a chemical or biological species related to the material of formula (β).

The substrate of claim 1 or 2, wherein the substrate is an organic or inorganic substrate.

The substrate of claim 1 or 2, wherein the substrate is made of a material selected from the group consisting of glass, silica, polycarbonate, polymethyl methacrylate, polystyrene, cycloolefin copolymer, and acrylonitrile polystyrene. .

5. The substrate according to claim 2, wherein the species being biological, it is chosen from the group consisting of a eukaryotic cell, a prokaryotic cell, a protein, a glycoprotein, an enzyme, an antigen, an antibody, DNA, RNA, a hormone, a living tissue, a protein or glycoprotein network.

6. Substrate according to claim 1 or 2, wherein at least one of the surfaces of the substrate comprises both the material of formula (α) and the material of formula (β).

The substrate of claim 6, wherein said at least one surface of the substrate is a structured surface and / or a surface having electrical functions.

8. Substrate according to claim 1 or 6, wherein the material of formula (β) is functionalized for the attachment thereto of a chemical or biological species.

9. Microsystem of chemical or biochemical analysis comprising a substrate according to any one of claims 1 to 8.

10. A biochip comprising a substrate according to any one of claims 1 to 8.

11. A method of locating a chemical or biological species on a substrate comprising the following steps:

(a) providing a substrate;

(b) depositing on at least one surface of the substrate a layer of a material of formula (α) SiO _x C _y : H where 0.7 <x <1.3 and 1.5 <y <2; (c) modifying by ultraviolet or plasma treatment at least one localized zone of the the

material of formula (α) deposited in step (b) in a material of formula (β) SiO _x iC _y i: H where 1.7 ≤ xl ≤ 2.5 and 0.1 <yl <0.4; and

The method of claim 11, wherein the substrate is an organic or inorganic substrate.

The method of claim 11, wherein the substrate is made of a material selected from the group consisting of glass, silica, polycarbonate, polymethyl methacrylate, polystyrene, cycloolefin copolymer, and polystyrene acrylonitrile.

14. The method of claim 11, wherein the layer of material of formula (α) is deposited on said surface of the substrate by plasma-enhanced chemical vapor deposition of a polysiloxane.

The process according to claim 13, wherein the polysiloxane is hexamethyldisiloxane or

Octamethylcyclotetrasiloxane, tetramethylcyclotetrasiloxane.

16. The method of claim 14, wherein the layer of material of formula (α) is deposited on said surface of the substrate at a thickness of 5 nm to 5 μm.

The method of claim 11, wherein the localized area is defined by positioning an absorbent mask around said at least one area during photochemical processing.

18. The method of claim 11, wherein the ultraviolet treatment is carried out in the presence of oxygen.

19. The method of claim 11, wherein step (d) is performed by immersing the substrate obtained in step (c) in said solution of the biological species.

20. The method of claim 11, wherein step (d) is carried out by dropwise deposition of said solution of the biological species on said localized area of material (β) obtained in step (c).

21. The method according to claim 11, wherein the species being biological, it is chosen from the group consisting of a eukaryotic cell, a prokaryotic cell, a protein, a glycoprotein, an enzyme, an antigen, an antibody, DNA, RNA, a hormone, a living tissue, a protein or glycoprotein network.

22. Use of a method according to any one of claims 11 to 21 for the manufacture of a biochip or a microsystem for chemical or biochemical analysis.

23. Use of a method according to any one of claims 11 to 21 for the location of glue on a hydrophilic zone.

24. Use of a method according to any one of claims 11 to 21 for locating drops of liquid for electrically connecting two surfaces.

25. Use of a method according to any one of claims 11 to 21 for locating magnetic elements for a localized deposition of magnets after evaporation of a drop of solution comprising said magnets.

26. Use of a method according to any one of claims 11 to 21 for depositing drops forming microlenses or deposition of liquid crystals in the field of optics.

A fuel cell comprising an electrolyte and electrodes, wherein the wettability of at least one of the electrodes is modified by a method comprising the steps of: (a) providing an electrode that serves as a substrate;

(c) modifying by ultraviolet or plasma treatment at least one localized zone of the layer of material of formula (α) deposited in step (b) in a material of formula (β) SiO _x iC _y i: H where 1, 7 <xl <2.5 and 0.1 <yl <0.4.

28. The cell of claim 27, wherein the cathode or anode has been plasma treated.

29. The cell of claim 28, wherein the plasma used is a helium plasma.

30. The cell of claim 28, wherein the plasma used is an oxygen plasma.

A method of manufacturing a fuel cell comprising an electrolyte and electrodes, wherein the wettability of at least one of the electrodes is modified by a method comprising the steps of:

(a) providing an electrode that serves as a substrate;

(b) depositing on at least one surface of the substrate a layer of a material of formula (α)

SiO _x C _y : H where 0.7 <x <1.3 and 1.5 <y <2; (c) modifying by ultraviolet or plasma treatment at least one localized zone of the layer of material of formula (α) deposited in step (b) in a material of formula (β) SiO _x iC _y i: H where 1, 7 ≤ xl ≤ 2.5 and 0.1 <yl <0.4.

32. The method of claim 31, wherein the cathode or anode has been plasma treated.

33. The method of claim 32, wherein the plasma used is a helium plasma.

34. The method of claim 32, wherein the plasma used is an oxygen plasma.